Molecules inhibiting a metabolic pathway involving the syk protein tyrosine kinase and method for identifying said molecules

ABSTRACT

The present invention relates to the C-13 molecule (methyl 2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}-benzoate) and to organic molecules functionally equivalent to the C-13 molecule, capable of inhibiting the binding of an antibody or antibody fragment with the human Syk protein tyrosine kinase, to the use of these molecules for the production of medicaments for the prevention or treatment of diseases dependent on metabolic pathways involving Syk, and also to a method for identifying such molecules.

The present invention relates to organic molecules capable of inhibitingthe binding of an antibody or antibody fragment with human Syk tyrosinekinase protein, the use of said molecules for producing medicinalproducts for the prevention and treatment of conditions dependent onmetabolic pathways involving Syk, and a method for identifying suchmolecules.

Syk (“Spleen tyrosine kinase”) Tyrosine Kinase protein (PKT) is acytoplasmic protein which is a key mediator in immunoreceptor-dependentsignalling in the cells involved in inflammation such as B lymphocytes,mast cells, macrophages and neutrophils. In mast cells and basophils,cross-linking of the FcεRI receptor (receptor with high affinity forimmunoglobulin E) with IgE and antigens induces phosphorylation of FcεRIITAM (“Immunoreceptor Tyrosine-based Activation Motif”) motifs so as toform a binding site for Syk which is then activated. The activated Sykprotein in turn phosphorylates numerous substrates, including LAT(“linker for activation of T cells”), SLP-76 (“Src homology 2 (SH2)domain-containing leukocyte protein of 76 kD”) and Vav adapter proteins,resulting in the activation of a plurality of signalling cascades, suchas those of PLC-γ (phospholipase Cγ), PI3K (“phosphatidylinositol3-kinase”), Erk (“extracellular signal-regulated kinase”), JNK (“c-junN-terminal kinase”) and p38 (see FIG. 9). These cascades eventually giverise to the degranulation, synthesis and release of lipid mediators andthe production and secretion of cytokines, chemokines and growth factorsby mast cells and basophils^(1,2).

Syk protein is thus recognised as a potential pharmaceutical target,particularly for the treatment of type I hypersensitivity reactionsincluding allergic rhinitis, urticaria, asthma and anaphylaxis due toits critical position upstream from immunoreceptor signalling complexesregulating the inflammatory response in leukocytes. The fact that Sykregulates FcεRI signalling positively³, particularly suggests that itcould be an excellent target for the treatment of allergic disorders.Furthermore, due to the central role thereof in FcεRI-dependentsignalling, interacting pharmaceutically with Syk could prove to be moreadvantageous than the conventional use of antihistamines or leukotrienereceptor agonists inhibiting a single step downstream from the complexcascades contributing to the acute and chronic symptoms associated withallergic conditions.

Pharmacological inhibitors of Syk kinase activity having a therapeuticpotential, such as, in particular, Syk-specific anti-senseoligonucleotides in the form of aerosols or small molecules interferingwith Syk activity such as ER-27139, BAY-613606, piceatannol and R112have already been developed^(1, 4). However, if multiple types of cellsexpressing Syk are considered, potential side effects associated withsystemic exposure of the immune system to medicinal products targetingthe Syk kinase domain need to be taken into consideration. Indeed, Sykprotein is widely distributed in various cell types, it is thusessential to account for the adverse effects of the inhibition thereofon varied physiological functions such as cell differentiation, adhesionand proliferation^(5, 6).

The inventors identified Syk protein inhibitors which act by preventingthe interaction thereof with the natural cellular partners thereofrather than by targeting the catalytic site thereof, particularlycompound C-13 and compounds 1 to 87 given in table 1.

The present invention relates to the molecule C-13 (methyl2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoate)having the formula

as a medicinal product for the prevention or treatment of a conditiondependent on a metabolic pathway involving Syk in humans or animals.

The present invention further relates to functionally equivalent organicmolecules to molecule C-13, binding with Syk tyrosine kinase protein andparticularly molecules capable of inhibiting by at least 5%, preferablyat least 10%, for example at least 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80 or 85% in vitro binding of

-   -   (i) antibody fragment G4G11 (SEQ ID No. 2), or    -   (ii) antibody fragment G4E4 (SEQ ID No. 3), or    -   (iii) an antibody or antibody fragment which binds with human        Syk tyrosine kinase protein on an epitope comprising at least        one of residues 65 to 74 of the amino acid sequence of human Syk        tyrosine kinase protein represented by the sequence SEQ ID No.        1, or    -   (iv) an antibody or antibody fragment which binds with human Syk        tyrosine kinase protein and inhibiting by at least 10% the        binding of antibody fragments G4G11 or G4E4 with human Syk        tyrosine kinase protein (SEQ ID No. 1)        with human Syk tyrosine kinase protein or with any of the        variants thereof in animals, for example murine Syk tyrosine        kinase protein wherein the sequence is illustrated in FIG. 8B        (SEQ ID No. 4), as a medicinal product for the prevention or        treatment of a condition dependent on a metabolic pathway        involving Syk in humans or animals.

The term “functionally equivalent molecule” refers to a molecule, forexample an organic molecule having a molecular weight between 50 and2500 Da, capable of resulting in the same effect in vitro in an intra orextracellular medium or in vivo, optionally with a different intensity,than a given molecule. In particular, within the scope of the presentinvention, the term “functionally equivalent molecule to molecule C-13”refers to a molecule capable of producing the same effect in vitro in anintra or extracellular medium or in vivo, optionally with a differentintensity, on human tyrosine kinase protein as represented by sequenceSEQ ID No. 1 (FIG. 8A) as molecule C-13. In particular, it refers tomolecules inhibiting the binding of Syk with another protein produced onthe Syk region comprising the SH2 domains thereof. More specifically,these molecules do not affect the kinase enzyme activity of Syk. Forexample, this consists of molecules capable of inhibiting theinteraction of Syk tyrosine kinase protein with antibody fragment G4G11(SEQ ID No. 2), antibody fragment G4E4 (SEQ ID No. 3), or an antibody orantibody fragment binding with the same epitope as antibody fragmentG4G11 or G4E4 on human Syk protein (SEQ ID No. 1).

The term “percentage of inhibition” of the binding of an antibody orantibody fragment with Syk protein, particularly refers to the ratio[(A−B)/(A×100)], where A consists of the intensity of a signalproportional to the quantity of an antibody or antibody fragment boundwith Syk protein in the absence of a molecule according to the inventionand B the intensity of the same signal in the presence of a moleculeaccording to the invention under the same conditions. The inhibition ofthe binding of an antibody or antibody fragments with Syk protein mayparticularly be demonstrated in vitro by an antibody displacement testbased on the ELISA technique as described for example in internationalapplication WO 2005106481. This test may be performed for exampleaccording to the protocol described in example 3-2) hereinafter.

The term “Syk variants in animals” refers to the genes of various animalspecies, for example mouse, rat, dog, cat or another mammal, coding fora protein having a strong sequence homology or identity with human Sykprotein as represented by the sequence SEQ ID No. 1 (see FIG. 8A), forexample a protein having at least 70, 75, 80, 85, 90 or 95% sequencehomology or identity with the sequence SEQ ID No. 1 of human Sykprotein, having the same tyrosine kinase activity and involved in thesame functional cascades as same, particularly in the functional cascadegiving rise to mast cell degranulation. It may particularly refer toorthologous genes, i.e. genes found in different organisms, havingevolved from the same ancestral gene following speciation events.

The present invention also relates to the pharmaceutically acceptablesalts, and if applicable, stereoisomers and racemates of C-13 or ofequivalent molecules according to the invention.

The term “pharmaceutically acceptable salts” refers to relativelynon-toxic inorganic and organic acid or basic addition salts preservingthe biological activity of the molecules according to the invention.Examples of pharmaceutically acceptable salts are particularly describedin S. M. Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci, 1977, 66:p. 1-19⁴⁰. The pharmaceutically acceptable addition salts of moleculesaccording to the invention may for example be hydrobromide,hydrochloride, sulphate, bisulphate, phosphate, nitrate, acetate,oxalate, valerate, oleate, palmitate, stearate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, naphthylate, mesylate, glucoheptanate,lactobionate, sulphamate, malonate, salicylate, propionate,methylenebis-b-hydroxynaphthoate, gentisic acid, isethionate,di-p-toluoyltartrate, methanesulphonate, ethane-sulphonate,benzenesulphonate, p-toluenesulphonate, cyclohexyl sulphamate andquinateslaurylsulphonate salts, and equivalents. Other pharmaceuticallyacceptable salts which may be suitable include metal salts, for examplepharmaceutically acceptable alkaline metal or alkaline-earth salts, suchas sodium, potassium, calcium or magnesium salts.

These pharmaceutically acceptable salts may be prepared in situ duringthe final molecule isolation and purification. Alternatively, the acidor basic addition salts may be prepared by reacting the purifiedmolecule separately in the acid or basic form thereof with a base or anorganic or inorganic acid and by isolating the salt formed. For example,a pharmaceutically acceptable acid addition salt may be prepared byreacting a molecule according to the invention with a suitable organicor inorganic acid (such as for example hydrobromic, hydrochloric,sulphuric, nitric, phosphoric, succinic, maleic, formic, acetic,propionic, fumaric, citric, tartaric, lactic, benzoic, salicylic,glutamic, aspartic, p-toluene-sulphonic, benzene-sulphonic,methane-sulphonic, ethane-sulphonic, hexanoic or naphthalene-sulphonicacids such as 2-naphthalene sulphonic acid), optionally in a suitablesolvent such as an organic solvent. A basic addition salt may, when asuitable acid group is present, be prepared by reacting a moleculeaccording to the invention with a suitable organic or inorganic base(for example triethyl-amine, ethanol-amine, triethanol-amine, choline,arginine, lysine or histidine), optionally in a suitable solvent such asan organic solvent. The salts thus generated may then be isolated bymeans of crystallisation and filtration.

In some embodiments, pharmaceutically acceptable salts are preferred inthat they provide the molecules according to the invention with superiorstability or solubility, facilitating the formulation thereof.

According to one particularly preferred embodiment, the organicmolecules according to the invention bind with Syk tyrosine kinaseprotein at a site located outside the catalytic domain thereof.

Also preferably, the organic molecules according to the invention have amolecular weight between 50 and 2500 Dalton, for example between 50 and2000 Da, between 50 and 1500 Da or between 50 and 1000 Da.

According to one particular embodiment, the molecules according to theinvention are capable of inhibiting, by at least 5%, preferably by atleast 10%, the binding of an antibody or antibody fragment binding withhuman Syk tyrosine kinase protein on an epitope comprising at least two,preferably at least 3, 4 or 5, for example 5, 6, 7, 8, 9 or 10 ofresidues 65 to 74 of the amino acid sequence of human Syk tyrosinekinase protein (SEQ ID No. 1), with human Syk tyrosine kinase. Accordingto one preferred embodiment, the antibody or antibody fragment bindswith human Syk tyrosine kinase protein on the same epitope as antibodyfragment G4G11 or antibody fragment G4E4 on human Syk tyrosine kinaseprotein, the sequence of which is illustrated by SEQ ID No. 1.

According to a further particular embodiment, the molecules according tothe invention are capable of inhibiting, by at least 5%, preferably byat least 10%, the binding of an antibody or antibody fragment whichbinds to with Syk tyrosine kinase protein and inhibits, by at least 15%,preferably by at least 20, 30, 40, 50, 60, 70 or 80%, the binding ofantibody fragments G4G11 or G4E4 with human Syk tyrosine kinase protein(SEQ ID No. 1), with human Syk tyrosine kinase protein.

Preferably, the molecules according to the invention bind with human Sykprotein on a three-dimensional cavity comprising the Arginine residuesituated in position 68 and the two glutamic acid residues situated inpositions 121 and 155 of the Syk protein, the sequence of which isillustrated by SEQ ID No. 1. Preferably still, the three-dimensionalcavity further comprises the Serine residue situated in position 9, theGlutamine residue situated in position 43, the Phenylalanine residuesituated in position 51, the Isoleucine residue situated in position 66,the Glutamate residues situated in position 67 and 69, the Leucineresidue situated in position 70, the Asparagine residue situated inposition 71, the Glycine residue situated in position 72, the Threonineresidue situated in position 73, the Tyrosine residue situated inposition 74 and the Alanine residue situated in position 75 of human Sykprotein, the sequence of which is illustrated by SEQ ID No. 1.

More preferably, the in vitro affinity, measured by the dissociationconstant (or Kd), of the molecules according to the invention for Sykprotein, is less than 100 more preferably, less than 50 and particularlypreferably, less than 25 μM. The affinity of the molecules according tothe invention for Syk protein is, for example, between 0.01 and 100 μM,between 0.1 and 50 μM or between 0.5 and 25 μM. The dissociationconstant of the molecules according to the invention with respect to Sykprotein may particularly be measured in vitro by means of fluorescencespectroscopy (or spectrofluorometry).

The present invention further relates to the use of a molecule accordingto the invention for producing a medicinal product for the prevention ortreatment of a condition dependent on a metabolic pathway involving Sykin humans or animals.

According to one particularly preferred embodiment, the molecules orsalts according to the invention are used for producing a medicinalproduct for the prevention or treatment of type I hypersensitivityreactions.

The term “hypersensitivity” refers to an unsuitable or excessive immuneresponse to an allergen, for example pollen, dust, animal hairs orcertain foods, with effects ranging from moderate allergic reaction(skin rash, rhinitis, conjunctivitis, etc.) to severe systemic reactionspotentially resulting in anaphylactic shock and potentiallylife-threatening in some cases. Immediate and delayed hypersensitivityreactions are classified in types I and IV respectively of theclassification defined by Gell and Coombs (Gell P G H, Coombs R R A,eds. Clinical Aspects of Immunology. 1st ed. Oxford, England: Blackwell;1963³⁹). According to this classification, “type I (or atopic oranaphylactic) hypersensitivity” is an immediate allergic reactionassociated with exposure to a specific antigen or allergen, for exampleby swallowing, inhalation, injection or direct contact, and thetriggering of immunoglobulin E (IgE) secretion by plasma cells. The IgEbinds with the Fc receptors found on the surface of tissue mast cellsand blood basophils. Subsequent exposure to the sensitised mast cellsand basophils to the same allergen gives rise to the degranulation ofthe cells having the corresponding IgE and the release of mediators suchas histamine, leukotriene or prostaglandins acting on the surroundingtissues, particularly giving rise to vasodilation and smooth musclecontraction. The reactions may be local or systemic and the symptomsvary from moderate irritation to sudden death due to anaphylactic shock.Examples of conditions caused by type I hypersensitivity includeallergic asthma, allergic conjunctivitis, allergic rhinitis (hay fever),anaphylaxis, angioedema, urticaria, eosinophilia, allergies toantibiotics such as penicillin or cephalosporin. “Type IIhypersensitivity” or “antibody-dependent immune response” is a reactiongenerally requiring from a few hours to one day, associated withinteractions between antibodies (IgG, IgM) and an antigen on the surfaceof the cells of the patient carrying this antigen, giving rise to thedestruction of these cells and the proliferation of B lymphocytes,producing antibodies against the antigen. “Type III hypersensitivity” or“immune complex disease” is a reaction developing over a number ofhours, days or weeks, associated with the presence of similar quantitiesof antibodies and antigens giving rise to the formation of immunecomplex not suitable for evacuation circulating in the vessels, thedeposition thereof on the walls of said vessels and giving rise to localor systemic inflammatory responses. “Type IV hypersensitivity” or“cell-mediated immunity” or “delayed hypersensitivity reaction” is animmune reaction generally requiring two to three days to develop and notassociated with an antibody response but with the formation of a complexbetween cells which express a major histocompatibility complex I or IIantigen and T lymphocytes giving rise to the release of lymphokinesand/or cytotoxicity mediated by T lymphocytes.

Preferably, the molecules or salts according to the invention are usedfor producing medicinal products for the prevention or treatment of typeI hypersensitivity reactions which inhibit IgE-dependent mast celldegranulation. More preferably, the molecules according to the inventionare capable of inhibiting by 50% in vitro mast cell degranulation, at aconcentration (IC50) between 1 ng/ml and 1 mg/ml, for example at aconcentration between 1 ng/ml and 500 μg/ml, between 1 ng/ml and 250μg/ml, between 1 ng/ml and 100 μg/ml, between 1 ng/ml and 50 μg/ml,between 1 ng/ml and 10 μg/ml, between 1 ng/ml and 5 μg/ml or between 1ng/ml and 2 μg/ml. Also preferably, a quantity between 1 nM and 1 mM,for example between 1 nM and 100 nM, between 10 nM and 100 nM or between1 nM and 10 nM, of a molecule according to the invention is capable ofinhibiting mast cell degranulation by 50% in vitro.

More preferably, the metabolic pathway involving Syk on which themolecules or salts according to the invention is a mast cell or basophilactivation pathway.

More preferably, the condition on which the molecules or salts accordingto the invention act is allergic asthma, allergic conjunctivitis,allergic rhinitis, anaphylaxis, angioedema, urticaria, eosinophilia oran allergy to an antibiotic.

According to one preferred embodiment, the molecules or salts accordingto the invention have no effect on the metabolic pathways involvinghuman Syk protein (SECT ID No. 1) other than those giving rise to mastcell degranulation and/or type I hypersensitivity reactions. Morepreferably, the molecules or salts according to the invention have noeffect on the antibody response following immunisation by athymus-dependent antigen or on Syk-dependent neutrophil recruitment.

Syk tyrosine kinase protein is also found on the surface of Blymphocytes, T lymphocytes, neutrophils, eosinophils, NK cells,platelets, erythrocytes, osteoclasts, epithelial cells or cancer cells.According to one alternative embodiment, the metabolic pathway involvingSyk on which the molecules or salts according to the invention act is aB lymphocyte, T lymphocyte, neutrophil, eosinophil, NK cell, platelet,erythrocyte, osteoclast, epithelial cell or cancer cell activationpathway. According to this embodiment, the condition on which themolecules or salts according to the invention act may thus be rheumatoidarthritis, an autoimmune disease, inflammation or cancer.

According to one particular embodiment, the molecules or salts accordingto the invention may be used in combination with another therapeuticmolecule. For example, it may consist of a therapeutic molecule alsoused for the prevention or treatment of a condition dependent on ametabolic pathway involving Syk or, on the other hand, a therapeuticmolecule used for the prevention or treatment of a condition notdependent on a metabolic pathway involving Syk. According to onepreferred embodiment, the molecules or salts according to the inventionare used in combination with a molecule used for the treatment ofallergy or type I hypersensitivity or for the treatment of the symptomsassociated therewith. More preferably, the molecules or salts accordingto the invention are used in combination with epinephrine (oradrenaline), an H1 antihistamine, for example diphenhydramine,meclizine, fluphenazine, perphenazine, prochlorperazine,trifluoperazine, acrivastine, astemizole, cetirizine, levocetirizine,fexofenadine, loratadine, desloratadine, mizolastine, azelastine,levocabastine, olopatadine, cromoglicate, nedocromil, a non-steroidalanti-inflammatory drug (NSAID) or a steroidal anti-inflammatory drug,for example cortisone, hydrocortisone (or cortisol), cortisone acetate,prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone,deoxycorticosterone acetate or aldosterone. According to a furtherpreferred embodiment, the molecules or salts according to the inventionare used in combination with an allergic desensitisation (oranti-allergic vaccination), i.e. a treatment based on regular increasingdoses of an allergen. According to one particular embodiment, themolecule according to the invention is not used in combination with aglucocorticoid receptor agonist.

The molecules or salts according to the invention may be administered byany administration route, particularly by the oral, sublingual, nasal,ocular, local, intravenous, intraperitoneal, subcutaneous routes, byaerosol or by inhalation.

The molecules or salts according to the invention may particularly beadministered to adult, child or newborn human patients. The molecules orsales according to the invention may also be administered to animalpatients, particularly mammals such as dogs, cats, rats, mice.

In particular, the molecules or salts according to the invention areadministered to a human patient at doses determined particularly on thebasis of the patient's condition, medical history and age, for exampledoses between 0.1 mg/kg and 200 mg/kg.

The present invention also relates to a therapeutic method for thetreatment or prevention of a condition dependent on a metabolic pathwayinvolving Syk in a human or animal patient comprising the administrationof a molecule according to the invention to the patient at doses,intervals and periods determined particularly on the basis of thepatients condition, medical history and age.

According to one particularly preferred embodiment, the moleculesaccording to the invention are selected from all the moleculesconsisting of C-13, molecules No. 1 to 87 given in table No. 1 and themolecules having any of the following formulas (I), (II), (III) or (IV):

where

-   -   R1 is an optionally substituted aromatic group, or an optionally        substituted heterocycle comprising at least one S, O or N atom;

R2 is an optionally substituted aromatic group, an optionallysubstituted heterocycle, an optionally saturated carbon chain,comprising an amine group, an optionally saturated carbon chaincomprising an optionally substituted aromatic group or an optionallysaturated carbon chain comprising an optionally substituted heterocyclecomprising at least one S, O or N atom;

R3 is an optionally substituted phenyl, 2-pyridinyl, 3-pyridinyl or4-pyridinyl group;

where

-   -   n=0 or 1; n′=0 or 1;    -   R4 is an optionally saturated carbon chain comprising 1 to 5        carbon atoms, optionally substituted with an aromatic group;    -   R5 is an optionally substituted aromatic group or an optionally        substituted amine group;    -   R6 is a hydrogen atom, alkoxy group, alkyl group or halogen;    -   R7 is a hydrogen atom, alkoxy group, alkyl group or halogen;    -   R8 is a hydrogen atom, alkoxy group, alkyl group or halogen;

where

-   -   m=0, 1 or 2;    -   R9 is a hydrogen atom and R10 is an optionally substituted        phenyl group, or R9 and R10 are part of the same optionally        substituted heterocycle, or R9 and R10 are part of the same        optionally substituted aromatic group;    -   R11 is a hydrogen atom, alkoxy group or alkyl group;    -   R12 is a hydrogen atom, alkoxy group or alkyl group;    -   R13 is a hydrogen atom or an alkyl or alkoxy group;    -   R14 is a hydrogen atom or an alkyl or alkoxy group;

where

-   -   A is an oxygen or sulphur atom;    -   R15 is an optionally saturated carbon chain comprising 1, 2 or 3        carbon atoms, optionally substituted by an optionally        substituted aromatic group, an optionally substituted        heterocycle or an amine group belonging to optionally        substituted heterocycle;    -   R16 is a hydrogen atom, halogen or alkoxy group;    -   R17 is a hydrogen atom, alkoxy group or acetoxy group.

According to one particular embodiment, the group R1 of molecules havingformula (I) is selected from the following groups:

-   -   a phenyl group, optionally substituted by an F or Cl atom, a        methyl or ethyl group, an N,N-dimethyl-sulphonamide or two        groups selected from the methyl, ethyl, hydroxy, methoxy or        ethoxy groups,    -   a group

-   -   a furan group optionally substituted by a methyl, ethyl,        hydroxyl, methoxy or ethoxy group,    -   a thiophene group optionally substituted by a methyl, ethyl,        hydroxy, methoxy or ethoxy group;        the group R2 of molecules having formula (I) is selected from        the following groups:    -   a group

-   -   where R21 and R22 are carbon atoms each belonging to an alkyl        chain comprising 1, 2 or 3 carbon atoms, or both belonging with        the nitrogen atom with which they are bound to the same        optionally saturated heterocycle also comprising an oxygen atom        or a second nitrogen atom,    -   a or a group

-   -   where R23 and R24 are carbon atoms each belonging to an alkyl        chain comprising 1, 2 or 3 carbon atoms, or both belonging with        the nitrogen atom with which they are bound to the same        optionally saturated heterocycle also comprising an oxygen atom        or a second nitrogen atom,    -   or a group

and the group R3 of molecules having formulas (I) is selected from thefollowing groups:

-   -   a non-substituted 2-pyridinyl, 3-pyridinyl or 4-pyridinyl group,    -   a phenyl group optionally substituted by a benzoxy group, and/or        by a hydroxyl group, and/or by a methyl group, and/or by an        ethyl group, and/or by a propyl group, and/or by one or two Br,        F or Cl atoms, and/or by one to three hydroxyl, methoxy or        ethoxy groups.

According to one particular embodiment, when the group R3 of moleculeshaving formula (I) is a phenyl group, the group R2 of molecules havingformula (I) is not an aromatic group or a heterocycle.

According to one particular embodiment, the group R4 of molecules havingformula (II) is an optionally saturated carbon chain comprising 1, 2 or3 carbon atoms; the group R5 is a phenyl group or a secondary amine growsubstituted by an optionally substituted phenyl group, or by a group

the group R6 of molecules having formula (II) is a hydrogen or chlorineatom or a methyl, ethyl, hydroxyl, methoxy or ethoxy group; the group R7of molecules having formula (II) is a hydrogen or chlorine atom or amethyl, ethyl, hydroxy, methoxy or ethoxy group; and the group R8 ofmolecules having formula (II) is a hydrogen or chlorine atom or amethyl, ethyl, hydroxy, methoxy or ethoxy group.

According to one particular embodiment,

-   -   the group R9 of molecules having formula (III) is a hydrogen        atom and the group R10 is an optionally substituted phenyl        group, or the groups R9 and R10 belong to the same optionally        substituted heterocycle comprising 2 nitrogen atoms and 4 carbon        atoms;    -   the group R11 of molecules having formula (III) is a hydrogen        atom or methyl, ethyl, hydroxy, methoxy or ethoxy group;    -   the group R12 of molecules having formula (III) is a hydrogen        atom or methyl, ethyl, hydroxy, methoxy or ethoxy group;    -   the group R13 of molecules having formula (III) is a hydrogen        atom or methyl, ethyl, hydroxy, methoxy or ethoxy group;    -   and the group R14 of molecules having formula (III) is a        hydrogen atom or methyl, ethyl, hydroxy, methoxy or ethoxy        group.

According to a further particular embodiment, the group R15 of moleculeshaving formula (IV) is a group

the group R16 of molecules having formula (IV) is a hydrogen or chlorineatom or a methyl, ethyl, hydroxy, methoxy or ethoxy group and the groupR17 of molecules having formula (IV) is a methyl, ethyl, hydroxy,methoxy, ethoxy, acetoxy, methoxycarbonyl or ethoxycarbonyl group.

According to one particular embodiment, the molecule according to theinvention is not

or if it is any of these molecules, it is not used in combination with aglucocorticoid agonist.

The term carbon chain refers to an organic chain having a linear orcyclic, optionally branched, chain formation of adjacent carbon atoms,connected by covalent bonds, as the network thereof. A carbon chainaccording to the present invention may for example be a linear chainformation of one to twenty, preferably 1 to 12, 1 to 10, 1 to 6, 1 to 5or 1 to 4 carbon atoms. In particular, it may consist of an alkyl group,i.e. derived from an alkane (linear or branched saturated hydrocarbonmolecule) due to the loss of a hydrogen atom, for example a methylgroup, an ethyl group, a linear or branched propyl group or a linear orbranched butyl group or a linear or branched unsaturated hydrocarbonchain, for example an ethenyl or ethynyl group. It is understood thatthe electrons of the outer layer (4 in number) of each carbon atomforming the carbon chain network are not involved in a covalent bondwith a further carbon atom or with a heteroatom are involved in acovalent bond with a hydrogen atom.

The term heteroatom refers to a non-metallic atom other than carbon orhydrogen, for example oxygen, nitrogen, sulphur, phosphorus or halogens.

The term aromatic or aryl group refers to an unsaturated cycle systemobserving Hackers aromaticity rule. For example, it may consist of aphenyl group (group derived from a benzene nucleus).

The term heterocycle refers to a cycle system wherein one or a pluralityof carbon atoms is replaced by a heteroatom such as, for example,oxygen, nitrogen or sulphur. It may in particular consist of aromaticheterocycles, such as pyrrole, thiophene, furan and pyridine or ofsaturated heterocycles, such as sugars, or uses. For example, aheterocycle according to the invention comprises 2 to 8 carbon atoms and1 to 4 heteroatoms, preferably it comprises 2, 3, 4 or 5 carbon atomsand 1, 2, 3 or 4 heteroatoms.

Each atom belonging to a carbon chain, aromatic group, cycle orheterocycle according to the present invention may be substituted via acovalent bond by one or a plurality of halogens, for example Fluorine,Chlorine, Iodine or Bromine, and/or by one or a plurality of organicgroups, for example one or a plurality of aromatic (such as a phenylgroup), cyclic, heterocyclic (such as a furan or thiophene group), alkyl(such as a methyl group, ethyl group, linear or branched propyl group ora linear or branched butyl group), alkoxy (such as a methoxy (OCH₃) orethoxy (OCH₂CH₃) group), carboxyl (such as a carboxy (COON) group),carbonyl (such as an acetoxy (OCOCH₃) or methoxycarbonyl (COOCH₃) orethoxycarbonyl (COOCH₂CH₃) group), primary, secondary or tertiary amine,amide (such as an acetamide group) or sulphonamide (such as anN,N-dimethyl-sulphonamide group) groups. It is understood that asaturated, unsaturated or aromatic cycle or heterocycle may be mergedwith a further cycle, for example by means of a single or double bondbetween two carbon atoms.

The present invention also relates to a pharmaceutical compositioncomprising a molecule according to the invention and a pharmacologicallyacceptable excipient.

According to one embodiment, the pharmaceutical composition according tothe invention also comprises a further therapeutic molecule. Forexample, it may consist of a therapeutic molecule also used for theprevention or treatment of a condition dependent on a metabolic pathwayinvolving Syk or, on the other hand, a therapeutic molecule used for theprevention or treatment of a condition not dependent on a metabolicpathway involving Syk. According to one particular embodiment, thepharmaceutical composition according to the invention does not comprisea glucocorticoid receptor agonist.

According to a further particular embodiment, the pharmaceuticalcomposition according to the invention may be used in combination withone or a plurality of further pharmaceutical compositions. For example,it may be pharmaceutical compositions also used for the prevention ortreatment of a condition dependent on a metabolic pathway involving Sykor, on the other hand, pharmaceutical compositions used for theprevention or treatment of a condition not dependent on a metabolicpathway involving Syk. According to this embodiment, the pharmaceuticalcomposition according to the invention and the further pharmaceuticalcomposition(s) may be administered simultaneously or in alternation, bythe same administration route or by different routes. According to oneparticular embodiment, the pharmaceutical composition according to theinvention is not used in combination with a glucocorticoid receptoragonist.

The present invention also relates to a method for identifying anorganic molecule having a molecular weight between 50 and 2500 Daltonbinding with Syk tyrosine kinase protein and capable of inhibiting by atleast 5%, preferably at least 10%, for example at least 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85% in vitro the binding of(i) antibody fragment G4G11 (SEQ ID No. 2), or (ii) antibody fragmentG4E4 (SEQ ID No. 3), or (iii) an antibody or antibody fragment whichbinds with human Syk tyrosine kinase protein on an epitope comprising atleast one of residues 65 to 74 of the amino acid sequence of human Syktyrosine kinase protein represented by the sequence SEQ ID No. 1, or(iv) an antibody or antibody fragment which binds with human Syktyrosine kinase protein and inhibits by at least 10% the binding ofantibody fragments G4G11 or G4E4 with human Syk tyrosine kinase protein(SEQ ID No. 1), to human Syk tyrosine kinase protein or to any of thevariants thereof in animals, comprising at least the following steps:

-   -   a) screening, from a bank of candidate organic molecules having        a molecular weight between 50 and 2500 Da, those liable to bind        with Syk protein on the three-dimensional binding cavity on the        Syk protein of a molecule selected from the molecules having        formula C-13, I, II, III, IV or 1 to 87 as illustrated above;    -   b) selecting from the molecules identified in a) those capable        of inhibiting by at least 5%, preferably at least 10%, for        example at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,        75, 80 or 85% in vitro the binding of the antibody or antibody        fragment (i), (ii), (iii) or (iv) with Syk protein.

According to one particularly preferred embodiment, the molecule fromstep a) selected from the molecules having formula C-13, I, II, III, IVor 1 to 87 is the molecule C-13, molecule (59) or molecule (61).

According to one preferred embodiment, the method according to theinvention comprises an additional step prior to step a) for identifyingthe three-dimensional binding cavity on the Syk protein of the moleculeselected from the molecules having the formula C-13, I, II, III, IV or 1to 87. This prior step may particularly be performed by means of “insilico docking”.

The term “in silico docking” or “molecular docking” or “virtual docking”refers to the use of a bioinformatics tool for predicting and modellingthe position of a ligand in a macromolecule. In particular, in silicodocking tools can be used to calculate the probability that a givenchemical compound will be able to dock with an active target protein,for example on a previously identified three-dimensional binding cavity.

According to one preferred embodiment, the method according to theinvention comprises an additional step c) for selecting from themolecules identified in b) those capable of inhibiting by 50% in vitroat mast cell degranulation to a concentration (IC50) between 1 ng/ml and1 mg/ml, for example at a concentration between 1 ng/ml and 500 μg/ml,between 1 ng/ml and 250 μg/ml, between 1 ng/ml and 100 μg/ml, between 1ng/ml and 50 μg/ml, between 1 ng/ml and 10 μg/ml, between 1 ng/ml and 5μg/ml or between 1 ng/ml and 2 μg/ml.

According to one particularly preferred embodiment of the uses andmethods described above, the molecule according to the invention isselected from the group consisting of the molecule C-13 and moleculesNo. 1 to 87 given in table 1.

These molecules were identified by the inventors within the scope of aproject following a previous study (Dauvillier et al., 2002⁷), duringwhich they expressed scFv (“single chain variable domain”) (or“intracellular antibodies” or “intrabodies”), G4G11 (SEC) ID No. 2) andG4E4 (SEC) ID No. 3) antibody fragments in a mast cell line. This studydemonstrated the inhibitory effects of these “intracellular antibodies”or “intrabodies” on the release of allergic mediators induced by FcεRIstimulation on the mast cell membrane. The scFv G4G11 and G4E4 antibodyfragments were isolated from a combinatory bank screened against arecombinant protein containing the SH2 domains of Syk and inter-domain Aregion separating same, i.e. a portion of Syk protein not comprising theSyk kinase domain⁸.

The ADA (“antibody displacement assay”) method is a method developed bythe inventors and described in WO2005106481, particularly foridentifying a ligand capable of selectively modulating a functionalcascade involving a target, comprising a first step for identifying anintracellular antibody capable of binding with the target and modulatingthe functional cascade in question, a second step for screening from abank of small organic molecules, ligands modulating the binding betweenthe target and the intracellular antibody potentially being performed invitro in an extracellular test, and a third step for identifying fromthe modulating ligands obtained in step 2, those capable of modulatingthe functional cascade in the cell.

The inventors suggested the theory whereby the antibody fragments G4G11and G4E4 bind on a Syk region interacting with one or more essentialpartners in the functional cascade giving rise to degranulation. Takinginto consideration the limits of the use of intracellular antibodies intherapy, such as the effective transfer of the gene encoding theantibody in target cells⁹, the inventors sought to isolate organicmolecules acting as functional mimics of the intrabody G4G11 andsuitable for easier use in therapy. To this end, they used the ADAmethod for screening a bank of 3000 small organic molecules andidentifying potential allergic response inhibitors.

Among the 3000 small organic molecules tested, the inventors identifiedthe small molecule C-13 (methyl2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoate)and demonstrated the ability thereof to modulate the interaction of theantibody fragment G4G11 or G4E4 with Syk in vitro and inhibit mast celldegranulation induced by FcεRI in vitro.

The inventors particularly demonstrated the fact that the compound C-13inhibits anaphylactic shock when administered orally and has promisinganti-allergic properties, illustrating the strong therapeutic potentialof medicinal product candidates isolated using the approach describedherein.

The inventors also demonstrated the fact that C-13 binds with Syk on anewly identified cavity situated between both SH2 domains andinter-domain A of Syk (FIG. 1). The binding cavity of C-13 forms aunique interaction zone which is specific to Syk, and does notcorrespond to a known binding side of physiological ligands of Syk suchas doubly phosphorylated ITAM peptide on tyrosine residues (FIG. 1A).The results obtained suggest that C-13 inhibits the interaction of Sykwith some of the macromolecular substrates thereof, either directly inthat C-13 occupies a surface whereon a partner of Syk could establishdirect contact, and/or by means of an allosteric effect.

The biochemical studies conducted on mast cells indeed demonstrated thatC-13 inhibits FcεR1-dependent phosphorylation of SLP-76 on tyrosineresidues contributing to the adapting function thereof for the bindingand/or stabilisation of Btk, PLC-γ and Vav with the macromolecularsignalling complex formed with LAT^(22, 28-30) (FIG. 2). This affectsthe phosphorylation and catalytic activity of Btk and PLC-γ renewal inthe vicinity of Syk and/or Btk for the complete phosphorylation thereofwhich is required for maintaining calcium flow and exocytosis³¹⁻³⁵.Indeed, C-13 inhibited early (β-hexosaminidase release) and delayed(TNF-α secretion) mast cell responses induced by aggregation on theFcεRI receptors with an estimated IC50 of 2 μM (FIG. 3).

Significantly, the oral administration of a single dose of C-13inhibited IgE-induced passive systemic anaphylaxis (PSA) with anestimated IC50 of 110 mg/kg (FIG. 4), confirming the promisinganti-allergic properties of this compound. On the contrary, a singleoral administration of 100 mg/kg of C-13 did not affect Syk-dependentneutrophil recruitment induced by thioglycollate in the peritonealcavity in the presence of Bordetella pertussis toxin (FIG. 5A).Furthermore, the inventors demonstrated that, despite the fact thatBCR-dependent B lymphocyte in vitro proliferation was inhibited in adose-dependent fashion by C-13 (FIG. 5B), the antibody responses of miceimmunised with a thymus-dependent antigen were not affected by the oraladministration of 150 mg/kg of C-13 (FIG. 5C). Therefore, the moleculeC-13 does not affect some responses dependent on Syk but not dependenton mast cells in vivo at administration doses and periods at which it isliable to inhibit a severe allergic response.

Taking into consideration the lack of an apparent toxic effect followingthe oral or local administration of C-13 over a period ranging from onehour to 12 days, C-13 may be considered as the potential first member ofa new family of Syk inhibitors suitable for oral administration andpharmacologically active molecules having an anti-inflammatory effect.The pharmaceutical molecule screening approach described hereinrepresents a generic platform wherein the initial use of antibodiesmakes it possible to detect the domains of the target molecule having atherapeutic potential, thus facilitating the design of chemicalmolecules (via in silico and/or in vitro screening) capable acting asfunctional antibody mimics and as potential protein-protein interactioninhibitors. Furthermore, the inventors demonstrated that these smallmolecules can induce the desired response in cell and animal models,supporting the concept in favour of the replacement of largemacromolecules that are difficult to administer by small organicmolecules suitable for oral administration.

The possible binding site with Syk was predicted in silico, guided bythe location of the epitope of G4G11. One candidate cavity situated nextto the epitope of G4G11, on the interface situated between the two SH2domains and the inter-domain binder of Syk and comprising the residuesSer 9, Gln 43, Phe 51, Ile 66, Glu 67, Arg 68, Glu 69, Leu 70, Asn 71,Gly 72, Thr 73, Tyr 74, Ala 75, Glu 121 and Glu 155 was thus identified(FIG. 10). Targeted mutagenesis experiments confirmed that the residuesArg 68, Glu 121 and Glu 155 of human Syk protein (SEQ ID No, 1) play asignificant role in interaction with C-13, the mutation of said residuessuppressing the inhibition caused by C-13, whereas the binding of scFvG4G11 is maintained (FIG. 1A, 10). These results tend to confirm thetheory whereby the binding cavity of C-13 or Syk is located in thevicinity of the binding site of the intrabody G4G11.

The inventors then performed virtual docking on a molecule bank toidentify candidate molecules having the best binding properties on saidthree-dimension cavity, and tested the ability of said candidatemolecules to inhibit the binding of scFv G4G11 with Syk. These moleculesare given in table 1 (see example 2). The ability of these molecules toinhibit mast cell degranulation in vitro was also tested. With moleculesNo. 59 and 61 in particular the concentration inhibiting mast celldegranulation by 50% in vitro is in the region of 5 μM (see FIG. 6).

The results given in the experimental part and particularly in table 1demonstrate that the molecules or salts according to the inventioninhibit type I hypersensitivity reactions, particularly IgE-dependentmast cell degranulation, and are also capable of interfering in vivowith passive cutaneous and systemic anaphylaxis in BALB/c mice.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Binding of C-13 with Syk in vitro. A. Chemical structure ofC-13. 3D structure of cavity predicted and validated for C-13 (or“Compound 13”) comprising the residues Ser 9, Gln 43, Phe 51, Arg 68,Glu 121 and Glu 155 (mesh representation); the G4G11 epitope comprisesthe residues 65 to 74 of human Syk protein represented by SEQ ID No. 1.B. Binding of scFv G4G11 fragment with Syk measured using ADA method inthe presence of C-13 (▪). Binding of C-13 with Syk measured usingfluorescence spectroscopy (o) (Kd=4.8±0.2 μM). C. Binding of G4G11 withSyk mutants with ADA method. Significant inhibition with C-13 versusDMF: **P<0.01.

FIG. 2. C-13 inhibits FcεRI-induced mast cell activation. A. Theimmunoblots produced on RBL-2H3 cell lysates were analysed withimmunoblots with the specified antibodies. The in vitro kinaseactivities of the Syk, Btk and Lyn immunoblots were examined. B. TheRBL-2H3 cell lysates and C. BMMCs were subjected to electrophoresis andthe proteins were analysed by means of immunoblot. These data arerepresentative of at least two experiments.

FIG. 3. C-13 inhibits FcεRI-dependent calcium release and degranulation.A. FACS analysis of IgE-dependent calcium flow in RBL-2H3 cells. B.Release of β-hexosaminidase in RBL-2H3 cells sensitised with IgE/DNP. C.Release of β-hexosaminidase and, D. titration of TNF-α in BMMCs cellssensitised with IgE/DNP; C-13: 3 μM. E. FACS analysis of FcεRI surfaceexpression in RBL-2H3 cells. (0=0.25% DMF), Significant inhibition withC-13 versus DMF: **P<0.01 and *P<0.05.

FIG. 4. In vivo studies on BALB/c mice. A-C. PSA response. A.Temperature progression, B. Evans blue extravasation quantification, CPhotograph representing Evans blue extravasation according to an oraladministration of 130 mg/kg of C-13, a non-relevant compound (IR) or thevector (T); Untreated animal (NT). The ears and tail of the micereceived the non-relevant compound (IR) or the vector alone (T) turned apronounced blue colour, those of the mice treated with C-13 turned alight blue colour and those of the untreated mice are normal in colourD. PCA response: Evans blue extravasation quantification. Significantinhibition with C-13 versus a non-relevant compound: **P<0.01; versusthe vehicle+DNP-KLH: *P<0.05.

FIG. 5. In vivo and in vitro effects of C-13 on neutrophils and Blymphocytes. A. Neutrophil recruitment in the peritoneal cavity afterinjecting thioglycollate or the vehicle in the presence of BordetellaPertussis toxin (where specified) (n=4). B. In vitro B lymphocyteproliferation induced by anti-IgM. C. Serum immunoglobulin concentration12 days after immunisation with TNP-KLH (n=4).

FIG. 6. Effect of molecules No. 59 (ref. Chembridge 7501888) and 61(ref. Chembridge 7722851) on RBL-2H3 cell degranulation. The level ofβ-hexosaminidase release in RBL-2H3 cells sensitised with IgE/DNP wasmeasured in the presence A. of molecule No. 59 and B. of Molecule No. 61at concentrations ranging from 0 to 40 μM.

FIG. 7. Amino acid sequences of intrabodies scFv A. G4G11 (SEQ ID No. 2)and B. G4E4 (SEQ ID No. 3).

FIG. 8. Amino acid sequence of human A. (SEQ ID No. 1) corresponding toaccession No. NP_(—)003168 (NCBI) and B. murine Syk proteincorresponding to accession No. NP_(—)035648 (NCBI). The residuesidentified as belonging to the three-dimensional binding cavity of C-13on human Syk protein (A) and the equivalent thereof on murine Sykprotein (B) are shown in bold type and underlined.

FIG. 9. Schematic representation of the metabolic pathways involving Syktyrosine kinase in mast cells.

FIG. 10. Toxicity test of C-13 on BMMC cells. The BMMC cells wereincubated at 37° C. in the presence of 2.5 μM or 5 μM of C-13, 0.25% DMFor Staurosporine. The percentages of live BMMC cells after 3 hours andafter 5 days were detected by means of double Annexin-V and PropidiumIodide labelling. The viability of BMMC cells incubated in the presenceof 2.5 μM or 5 μM of C-13 or 0.25% DMF was not affected to noteworthydegree after 3 hours (78%, 74% and 79% respectively) or five days (62,56 and 57%, respectively). On the other hand, the viability of BMMCcells treated under the same conditions with Staurosporine was reducedto a level of 16% after only 3 hours.

EXAMPLES Example 1 Identification of Molecule C-13 Potentially Suitablefor Oral Administration to Prevent Anaphylactic Shock

1) Identification of Compound 13 (C-13) and the Binding Cavity thereofon Syk

The inventors developed the ADA (Antigen Displacement Assay) method toidentify small molecules capable of displacing the association with scFvG4G11 with Syk. Of the members of a bank of 3000 chemical molecules, 15small molecules proved to be capable of competing with the binding ofscFv G4G11 with Syk, and of these compounds, that hereinafter referredto as C-13 (FIG. 1A) displayed the best inhibition potential with anestimated IC50 of 4 μM (FIG. 1B). This result is in line with thedissociation constant value (or Kd) equal to 4.8 μM obtained bymeasuring by means of fluorescence spectroscopy (or spectrofluorometry)the in vitro affinity of C-13 for Syk (FIG. 1B). To understand the modeof action of C-13, the inventors firstly identified the binding site ofG4G11 using the SPOT method¹⁶. An epitope located on the N-terminal SH2domain of Syk, comprising amino acids 65-74 and 100% preserved in mouse,rat and human sequences, was identified.

On the basis of this information and the known 3D structure of thepeptide complex formed by the SH2 domains of Syk and ITAM motifs¹⁷, theinventors used computing approaches to locate a putative binding sitefor C-13. A candidate cavity comprising the residues Ser 9, Gln 43, Phe51, Ile 66, Glu 67, Arg 68, Glu 69, Leu 70, Asn 71, Gly 72, Thr 73, Tyr74, Ala 75, Glu 121 and Glu 155 of human Syk protein (see FIG. 8A, SEQID No. 1) and situated in the vicinity of the G4G11 epitope wasidentified (FIG. 1C). A structural analysis specified that the residuesSer 9, Gln 43, Phe 51, Arg 68, Glu 121 and Glu 155 could be involved inthe binding of the ligand, and could be mutated without impairing the 3Dstructure of the protein. To validate the cavity in more detail, thesesix amino acids were mutated individually and the Syk mutants weresubjected to the ADA test. The residues Arg 68, Glu 121 and Glu 155proved to have a significant role in the interaction with the smallmolecule, given that the mutation thereof cancelled the inhibitioncaused by C-13, whereas the binding of scFv G4G11 was maintained (FIG.10). These data confirmed the fact that the binding cavity of C-13 onSyk is located in the vicinity of the binding site of G4G11.

2) FcεRI-Induced Mast Cell Activation

To examine the functional similarities with G4G11, the inventorsexplored the biological effects of C-13 on mast cell activation. Theincubation of RBL-2H3 cells with 0-13 did not affect the phosphorylationand FcεRI-induced kinase activity of Syk (FIG. 2A) and, accordingly, theoverall level of tyrosine phosphorylation of all the cell proteins knownas being essentially Syk-dependent was normal (FIG. 2B, C). Similarly tothe intrabody G4G11, C-13 inhibited the phosphorylation andFcεRI-induced kinase activity of Btk (FIG. 2A) and the phosphorylationof PLC-γ1 and PLC-γ2, the two PLC-γ isoforms expressed in mast cells(FIG. 2A, C). PTK Lyn phosphorylates both Syk and Btk giving rise to thecomplete activation thereof and the subsequent phosphorylation ofPLC-γ¹⁸. Given that C-13 did not affect FcεRI-dependent Lyn activation(FIG. 2A), it can be concluded that the reduction of the level of Btkand PLC-γ phosphorylation could be due to a defect with respect to thecorrect location thereof in the vicinity of the upstream PTK.

3) Fyn- and Lyn-Dependent Signalling Cascade Analysis

In mast cells, the signalling cascade Fyn/Gab2/PI3K gives rise to theactivation of PI3K and the generation of PI-3,4,5-P3 recruiting a numberof proteins containing a pleckstrin homology domain (PH), including Btkand PLC-γ on the plasma membrane¹⁹. The analysis of the phosphorylationof Akt, a PI3K activity marker, indicated that C-13 did not affect theFyn-dependent cascade (FIG. 2B, C), suggesting that the reduced level ofphosphorylation of Btk and PLC-γ was not due to a defect on the membranelocation thereof, known as being an essential factor in the increase incalcium flows²⁰.

Btk and PLC-γ recruitment on the membrane also requires the canonicalsignalling cascade Lyn/Syk/LAT/SLP-76. The phosphorylation of LAT by Sykgives rise to the translocation of SLP-76 to the complex organised byLAT²¹, where SLP-76 is co-located with Syk²². This location enables Sykto phosphorylate N-terminal tyrosines of SLP-76²³ which become bindingsites for Vav, Nck and Btk. LAT and SLP-76 (via the proline-rich domainthereof recruiting PLC-γ) interact to locate PLC-γ on said membranecomplex, enabling the phosphorylation and activation of PLC-γ by Btk²⁴and/or Syk²⁵. The use of phospho-specific antibodies demonstrated thatC-13 inhibits the phosphorylation of SLP-76, but increases thephosphorylation of LAT in a dose-dependent fashion (FIG. 2A). Theinventors suggested the theory whereby the inhibition of SLP-76phosphorylation could enable a larger quantity of LAT to interact withSyk, thus causing an increase in the phosphorylation level thereof.These results demonstrate that the reduction in SLP-76 phosphorylationwas not due to a defect in terms of the recruitment thereof to LAT, andresulted in a co-location defect of Btk and, to a lesser extent, that ofVav with SLP-76 (FIG. 2A). Nevertheless, Vav phosphorylation known to beindependent from the recruitment thereof to SLP-76²⁶ was not inhibited(FIG. 2A).

4) MAPK Activation

The association of SLP-76 with Vav and/or Nck plays a role in optimalMAP kinase activation in mast cells²⁷. The inventors demonstrated thatC-13 affects MAP kinase activation slightly (evaluated via thephosphorylation level thereof): a high C-13 concentration reduces thephosphorylation level ERK1/2, whereas the phosphorylation levels of p38and JNK remain normal (FIG. 2B, C).

5) Calcium Flow and Degranulation

Binding of Btk and Vav with SLP-76 is critical for regulating PLC-γactivity on the membrane, calcium mobilisation and granuleexocytosis^(27, 28). The inventors demonstrated that the association ofPLC-γ with LAT was inhibited by C-13 in a dose-dependent fashion (FIG.2A). Consistently with the defect in terms of PLC-γ1 and PLC-γ2phosphorylation, the mast cells showed a reduced calcium flow range inresponse to FcεRI binding (FIG. 3A), and early and delayed FcεRI-inducedallergic responses in BMMC (“bone marrow derived mast cell”) cells andin the RBL-2H3 cell line are also weakened in a dose-dependent fashion,based on the measurement of β-hexosaminidase release and TNF-α secretion(FIG. 3B, C, D). The results also demonstrated that C-13 had no toxiceffect on mast cells. Indeed, ionomycin-induced degranulation (FIG. 3B),or BMMC cell viability (see FIG. 10) were not detectably affected bytreatment with C-13. Furthermore, the defects observed in terms of mastcell activation are not due to a reduced level of FcεRI surfaceexpression, flow cytometry analysis indicating that the cells incubatedwith C-13 express similar levels of FcεRI to those of control cells(FIG. 3E).

6) Passive Systemic (PSA) and Cutaneous Anaphylaxis (PCA)

Finally, to extend these observations to mast cell functions in vivo,the inventors tested the effects of C-13 on passive systemic (PSA) andcutaneous anaphylaxis (PCA) induced in BALB/c mice by administeringDNP-specific IgE molecules followed by intravenous stimulation withDNP-KLH hapten. This mimics systemic anaphylaxis as demonstrated by theimmediate cardiopulmonary changes and the increase in vascularpermeability. The intensity of systemic anaphylaxis was determined bymeasuring both the drop in body temperature and the increase in vascularpermeability following antigen administration. After the oraladministration of C-13 (and prior to antigen stimulation), the animalsappeared to be healthy with no obvious sign of toxicity. Theadministration of a single oral dose of 100 mg/kg of C-13 inhibitedhypothermia and accelerated the recovery of the animals (FIG. 4A). Onthe basis of the Evans blue extravasation quantification, it wasdetermined that C-13 inhibits the increase in vascular permeability withan estimated IC50 of 110 mg/kg (FIGS. 4B and 4C). C-13 also demonstratedan inhibitory effect on PCA with an estimated IC50 of 25 μM (FIG. 4D).

Example 2 Identification Using Binding Cavity of C-13 of FurtherPotential Mast Cell Degranulation Inhibitors

Using the binding cavity identified in example 1-1), virtual dockingscreening was conducted on a set of 350,000 molecules contained in theChemBridge Corporation (San Diego, USA) chemical bank to identify the1000 molecules displaying the best binding properties in said cavity.

The ADA method was then applied to each of these 1000 molecules tomeasure the ability thereof to inhibit the binding of scFv G4G11 withSyk. The 87 molecules having the best inhibition rate (between 11 and86.5%) are given in table 1.

These 87 molecules were also tested in vitro to assess the abilitythereof to inhibit RBL-2H3 cell degranulation (see table 1). The twomolecules exhibiting the best potential (molecules No. 59 and 61) weretested at various concentrations on RBL-2H3 cells to assess theconcentration inhibiting degranulation by 50% in more detail (see FIG.6).

Finally, the in vitro affinity of the molecule C-13 and some of the 87molecules mentioned above for Syk protein was measured by means offluorescence spectroscopy (or spectrofluorometry) and is expressed bythe dissociation constant (or Kd) in μmole/litre (μM).

TABLE 1 C-13 and the 87 molecules identified using C-13 and the antibodyfragment G4G11 CB In vit. Degran. Structure ref. Name Rank inhib. IC50Kd No. Gp

6197026 methyl 2-{5- [(3-benzyl-4- oxo-2-thioxo- 1,3- thiazolidin-5-ylidene) methyl]-2- furyl} benzoate — 81% 1 μg/ml 4.8 μM C-13 —

6752784 4-(4-chloro benzoyl)-3- hydroxy-5-(3- phenoxy phenyl)-1-(3-pyridinyl methyl)-1,5- dihydro-2H- pyrrol-2-one 611 86.5 >10 μg/ml 5 μM 1 I

6670340 5-(2,4- dimethoxy phenyl)-3- hydroxy-1-[3- (1H-imidazol-1-yl)propyl]-4- (2-thienyl carbonyl)-1,5- dihydro-2H- pyrrol-2-one 79281 >10 μg/ml 16.3 μM  2 I

6422575 {4-bromo-2- [3-(ethoxy carbonyl)-2- méthyl-5-oxo- 4,5-dihydro-1H- indeno[1,2- b]pyridin-4- yl]phenoxy} acetic acid 706 81 >10 μg/ml6.2 μM  3 —

6882059 3-hydroxy-5- (3-methoxy phenyl)-4-(4- méthyl benzoyl)-1-[3- (4-morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 243 79.5 >10 μM 9.4μM  4 I

7111786 4-benzoyl-5- (2,5- dimethoxy phenyl)-3- hydroxy-1-[3-(1H-imidazol- 1-yl)propyl]- 1,5-dihydro- 2H-pyrrol-2- one 557 77.5 >10μg/ml —  5 I

6203863 ethyl 4-[3- benzoyl-2- (2,4- dimethoxy phenyl)-4- hydroxy-5-oxo-2,5- dihydro-1H- pyrrol-1-yl] benzoate 423 73.5 >10 μg/ml 6.1 μM  6I

7347627 4-(2,5- dimethyl benzoyl)-3- hydroxy-5-(2- methoxy phenyl)-1-[2-(4- morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 775 72 >10 μg/ml — 7 I

7489416 5-(3-bromo-4- hydroxy-5- methoxy phenyl)-3- hydroxy-1-(2-phenylethyl)- 4-(2-thienyl carbonyl)-1,5- dihydro-2H- pyrrol-2-one 64871 >10 μg/ml —  8 I

6719738 4-(4-fluoro benzoyl)-3- hydroxy-5-(3- phenoxy phenyl)-1-(3-pyridinyl methyl)-1,5- dihydro-2H- pyrrol-2-one 977 71 >10 μg/ml —  9 I

6650234 ethyl 2-[3-(4- fluoro- benzoyl)- 4-hydroxy-2- (4-methylphenyl)-5-oxo- 2,5-dihydro- 1H-pyrrol-1- yl]-4-methyl- 1,3-thiazole-5-carboxylate 946 71 ~10 μg/ml 6.3 μM 10 I

6652639 5-(2,5- dimethoxy phenyl)-3- hydroxy-4-(4- methyl benzoyl)-1-(3-pyridinyl methyl)-1,5- dihydro-2H- pyrrol-2-one 829 70.5 >10 μg/ml — 11I

6673225 ethyl 2-[3- benzoyl-4- hydroxy-2-(4- methoxy phenyl)-5-oxo-2,5-dihydro- 1H-pyrrol-1- yl]-4-methyl- 1,3-thiazole-5- carboxylate 30169 >10 μg/ml — 12 I

6800873 3-[2-(2,4- dimethoxy phenyl)-2- oxoethyl]-3- hydroxy-1-(1-naphthyl methyl)-1,3- dihydro-2H- indol-2-one 250 67.5 >10 μg/ml 4.6 μM13 —

6879058 3-hydroxy-4- (4-methoxy-2- methyl benzoyl)-1-[2- (4-morpholinyl) ethyl]-5-(3- pyridinyl)-1,5- dihydro-2H- pyrrol-2-one 75867.5 >10 μg/ml — 14 I

6282824 2-methoxy-N- (4-{4-methyl- 5-[(2-oxo-2- phenylethyl)thio]-4H-1,2, 4-triazol-3- yl}phenyl) benzamide 905 67.5 ~2.5 μg/ml 5.6μM 15 II

6750319 methyl 2-[3- benzoyl-4- hydroxy-2-(4- méthyl phenyl)-5-oxo-2,5-dihydro- 1H-pyrrol-1- yl]-4-methyl- 1,3-thiazole-5- carboxyiate 85067 >10 μg/ml — 16 I

6474819 4-[(4-benzyl- 1-piperidinyl) methyl]-N-(2- methoxy-5-methylphenyl) benzamide 954 63 >10 μg/ml 17.9 μM 17 —

6498669 4-{[N-[(4- methoxy phenyl) sulphonyl]-N- (2-phenyl ethyl)glycyl]amino} benzamide 808 63 >10 μg/ml 0.8 μM 18 III

6651552 4-(4-fluoro benzoyl)-3- hydroxy-5-(4- isopropyl phenyl)-1-[3-(4- morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 100 60 >10 μg/ml— 19 I

6453860 N,N′-1,5- naphthalene- diylbis[2-(3- methyl- phenoxy) acetamide]145 60 >10 μg/ml — 20 —

6853966 N-[4-({[4- (acetyl amino) phenyl] sulphonyl} amino)-2,5-dimethoxy phenyl] benzamide 11 58 >10 μg/ml — 21 —

6866968 7,7-dimethyl- 1-(4-methyl phenyl)-2,5- dioxo-N- (2,2,6,6-tetramethyl-4- piperidinyl)- 1,2,5,6,7,8- hexahydro-3- quinolinecarboxamide 255 57.5 >10 μg/ml — 22 —

7938324 4-(benzyl{[1- phenyl-3-(2- thienyl)-1H- pyrazol-4- yl]methyl}amino)-4-oxo butanoic acid 795 57 >10 μg/ml — 23 _—

6905988 4-(4-fluoro benzoyl)-3- hydroxy-5-(3- methoxy phenyl)-1-[3- (4-morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 843 57 >10 μg/ml — 24I

6885782 4-(4-chloro benzoyl)-3- hydroxy-1-[3- (4- morpholinyl)propyl]-5-(3, 4,5-trimethoxy phenyl)-1,5- dihydro-2H- pyrrol-2-one 24956.5 >10 μg/ml — 25 I

6663684 4-benzoyl-3- hydroxy-5-(4- isopropyl phenyl)-1 -[2- (4-morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 530 56.5 >10 μg/ml —26 I

6672500 4-(4-chloro benzoyl)-5- (3,4- dimethoxy phenyl)-3- hydroxy-1-[2-(4- morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 194 55 >10 μg/ml —27 I

7721949 1-[2-(diethyl amino)ethyl]- 5-(2,5- dimethoxy phenyl)-3-hydroxy-4-(4- methyl benzoyl)-1,5- dihydro-2H- pyrrol-2-one 139 54 >10μg/ml — 28 I

7966545 2-fluoro-N- [(5- {[(4-oxo-3,4- dihydro-2- quinazolinyl)methyl]thio}- 4-phényl-4H- 1,2,4-triazol- 3-yl)methyl] benzamide 77353.5 ~10 μg/ml 6.7 μM 29 —

7437580 2-{[4-(1,3- dioxo-1,3- dihydro-2H- isoindol-2- yl)butanoyl]amino}-N- (tetrahydro-2- furanyl methyl)-5,6- dihydro-4H- cyclopenta[b]thiophene-3- carboxamide 222 51 >10 μg/ml — 30 —

6654239 5-(3,4- dimethoxy phenyl)-4-(4- fluoro benzoyl)-3- hydroxy-1-[2-(4- morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 343 50 >10 μg/ml —31 I

6670570 4-({4- hydroxy- 1-[2-(4- morpholinyl) ethyl]-5-oxo-2-phenyl-2,5- dihydro-1H- pyrrol-3-yl} carbonyl)- N,N-dimethyl benzenesulphonamide 926 50 >10 μg/ml — 32 I

6670673 5-(2,4- dimethoxy phenyl)-4-(4- fluoro- benzoyl)- 3-hydroxy-1-[2-(4- morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 742 50 >10μg/ml — 33 I

6670747 3-hydroxy-5- (4-methoxy phenyl)-4-(4- methyl benzoyl)-1-[2- (4-morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 413 50 >10 μg/ml — 34I

6671401 5-(3,4- diméthoxy phenyl)-4-(2- furoyl)-3- hydroxy-1-[2- (4-morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 954 50 >10 μg/ml — 35I

6672500 4-(4-chloro benzoyl)-5- (3,4- dimethoxy phenyl)-3- hydroxy-1-[2-(4- morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 194 50 >10 μg/ml —36 I

6673225 ethyl 2-[3- benzoyl-4- hydroxy-2-(4- methoxy phenyl)-5-oxo-2,5-dihydro- 1H-pyrrol-1- yl]-4-methyl- 1,3-thiazole-5- carboxylate 12450 >10 μg/ml — 37 I

6677533 5-(3,4- dimethoxy phenyl)-3- hydroxy-1-[2- (4- morpholinyl)ethyl]-4-(2- thienyl carbonyl)-1,5- dihydro-2H- pyrrol-2-one 409 50 >10μg/ml — 38 I

6683618 4-(4-chloro benzoyl)-3- hydroxy-5-(4- methoxy phenyl)-1-[2- (4-morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 808 50 >10 μg/ml — 39I

6417902 N-[2-(4- benzyl-1- piperazinyl)-2- oxoethyl]-N- (3,5-dimethylphenyl) benzene sulphonamide 897 49.5 >10 μg/ml — 40 III

6437157 1-methyl-2-(4- methyl phenyl)-2- oxoethyl 2-(3- chloro-4- methylphenyl)-1,3- dioxo-5- isoindoline carboxylate 871 49.5 >10 μg/ml — 41 —

6465972 N~2~-[(3,4- dimethoxy phenyl) sulphonyl]- N~1~-(2- methoxy-5-methyl- phenyl)- N~2~-(4- methylphenyl) glycinamide 926 49 >10 μg/ml —42 III

7723671 5-(2,5- dimethoxy phenyl)-4-(4- fluoro benzoyl)-3- hydroxy-1-[3-(4- morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 34 48.5 >10 μg/ml— 43 I

6648368 4-(4- chloro- benzoyl)- 5-(2- fluorophenyl)- 3-hydroxy-1- [3-(4-morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 465 48.5 >10 μg/ml —44 I

6656195 4-(4-fluoro benzoyl)-3- hydroxy-5-(4- isopropyl phenyl)-1-[2-(4- morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 613 48.5 >10 μg/ml— 45 I

7778331 4-{[2-(4- morpholinyl) ethyl]amino}- 3-(4- morpholinylsulphonyl)-N- phenyl- benzamide 998 48 >10 μg/ml 3 μM 46 —

6994060 2-chloro-N- {4-[4-methyl- 5-({2-oxo-2- [(tetrahydro- 2-furanylmethyl) amino] ethyl}thio)- 4H-1,2,4- triazol-3-yl] phenyl} benzamide623 47.5 >10 μg/ml — 47 II

6458830 N-[2-(4- benzyl-1- piperazinyl)-2- oxoethyl]-N- (3,4-dimethylphenyl)-4- methyl benzene sulphonamide 837 47 >10 μg/ml — 48 III

7524107 2-[(4-{[(4- isopropyl phenoxy) acetyl]amino}- 3-methyl benzoyl)amino] benzoic acid 789 46.5 ~10 μg/ml 5.5 μM 49 —

6661524 4-({2-(3,4- dichloro phenyl)-1-[3- (dimethyl amino)propyl]-4-hydroxy-5- oxo-2,5- dihydro-1H- pyrrol-3-yl} carbonyl)- N,N-dimethylbenzene sulphonamide 428 46.5 >10 μg/ml — 50 I

7739436 4-(1,3- benzodioxol- 5-yl carbonyl)- 1-[3-(diethylamino)propyl]- 3-hydroxy-5- (3-pyridinyl)- 1,5-dihydro- 2H-pyrrol-2- one976 46 >10 μg/ml — 51 I

6881804 1-[2-(dimethyl amino)ethyl]- 3-hydroxy-4- (5-methyl-2-furoyl)-5- (3,4,5- trimethoxy phenyl)-1,5- dihydro-2H- pyrrol-2-one 23046 >10 μg/ml — 52 I

6907921 3-hydroxy-5- (3-methoxy phenyl)-4-(5- methyl-2- furoyl)-1-[2-(4-morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 609 45.5 >10 μg/ml —53 I

7495334 ethyl 4-({[(5,6- di-2-furyl- 1,2,4-triazin-3- yl)thio]acetyl}amino) benzoate 380 43 ~5 μg/ml 1.6 μM 54 —

7509862 ethyl 5-cyano- 4-(2-furyl)-6- ({2-[(3- methoxy phenyl) amino]-2-oxoethyl} thio)-2-phenyl- 1,4-dihydro-3- pyridine carboxylate 170 42.5~5 μg/ml — 55 —

7325385 5-(2,3- dimethoxy phenyl)-4- (2,5-dimethyl benzoyl)-3-hydroxy-1-[3- (4- morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 6542 ~10 μg/ml — 56 I

6669449 3-(6-amino-5- cyano-3- phenyl-1,4- dihydro pyrano[2,3-c]pyrazol-4- yl)phenyl 2- furoate 140 42 >10 μg/ml — 57 —

7348779 1,4-bis [(mesityloxy) acetyl] piperazine 209 40 ~10 μg/ml — 58 —

7501888 N-(4-chloro pheny)-2-{[4- (2-phenyl ethyl)-5- (3,4,5- trimethoxyphenyl)-4H- 1,2,4-triazol- 3-yl]thio} acetamide 544 40 ~2 μg/ml 8.2 μM59 —

6946138 {[3-(ethoxy carbonyl)-2- phenyl-1- benzofuran-5- yl]oxy}(phenyl) acetic acid 298 40 >10 μg/ml — 60 —

7722851 ethyl 4-({[1- (4-chloro phenyl)-5- oxo-3-(3- pyridinylmethyl)-2- thioxo-4- imidazolidinyl] acetyl} amino) benzoate 638 39.5 ~2μg/ml 21.8 μM 61 IV

7517583 5,5′-oxybis [2- (tetra hydro-2- furanyl methyl)-1H- isoindole-1,3(2H)-dione] 912 38.5 >10 μg/ml — 62 —

6634701 7-acetyl-6-[3- (benzyloxy) phenyl]-3- (methylthio)- 6,7-dihydro[1,2,4] triazino[5,6- d][3,1] benzoxazepine 934 38.5 ~5 μg/ml — 63 —

7726450 3-hydroxy-1- [3-(1H- imidazol-1- yl)propyl]-4- [(7-methoxy-1-benzofuran- 2-yl) carbonyl]-5- (2-pyridinyl)- 1,5-dihydro-2H-pyrrol-2- one 472 37.5 >10 μg/ml — 64 I

6662088 4-{[4-hydroxy- 1-[3-(4- morpholinyl) propyl]-5-oxo- 2-(3-pyridinyl)- 2,5-dihydro- 1H-pyrrol-3- yl]carbonyl}- N,N-dimethylbenzene sulphonamide 156 37 >10 μg/ml — 65 I

7752193 N-{1-[4-allyl- 5-({2-[(3- methoxy phenyl) amino]-2- oxoethyl}thio)-4H-1,2,4- triazol-3- yl]ethyl} benzamide 248 35 ~5 μg/ml 8 μM 66 —

7238569 N-(2-hydroxy- 1,1-dimethyl- ethyl)-5-{4-[(3- hydroxyphenyl)amino]-1- phthalazinyl}- 2-methyl benzene sulphonamide 749 32 >10 μg/ml— 67 —

7724000 4-(1,3- benzodioxol- 5-ylcarbonyl)- 5-(2- fluorophenyl)-3-hydroxy-1- [3-(4- morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one303 30 ~10 μg/ml — 68 I

7443270 N-(2,4- dimethoxy- phenyl)- 2-{[3-(2- furylmethyl)-4- oxo-3,4-dihydro-2- quinazolinyl] thio} acetamide 895 30 ~5 μg/ml — 69 —

7245019 N-[(2-hydroxy- 7-méthyl-3- quinolinyl) methyl]-3- methoxy-N-(2-methoxy phenyl) benzamide 608 30 >10 μg/ml — 70 —

6909597 4-(4- chlorobenzoyl)- 3-hydroxy-5- (3-methoxy phenyl)-1-[2- (4-morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 790 29 >10 μg/ml — 71I

7661882 2-(4-methoxy phenoxy)-N- [2-methyl-5- (3-methyl-4- oxo-3,4-dihydro-1- phthalazinyl) benzyl] acetamide 941 28 ~10 μg/ml — 72 —

7667791 N-{4-[({[4-(4- methoxy phenyl) tetrahydro-2H- pyran-4-yl]methyl} amino) carbonyl] phenyl}-2- furamide 797 28 >10 μg/ml — 73 —

6891745 4-benzoyl-5- (2,3- dimethoxy phenyl)-3- hydroxy-1 -[3- (4-morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 115 27.5 >10 μg/ml —74 I

7783660 isopropyl 3- ({[(4-allyl-5- {[(3-methyl benzoyl) amino]methyl}-4H- 1,2,4-triazol-3- yl)thio]acetyl} amino) benzoate 266 27 ~5μg/ml 4.8 μM 75 II

7653478 2-{5-[1-(4- morpholinyl) cyclohexyl]- 1H-tetrazol-1- yl}ethyl 1-naphthyl carbamate 365 27 >10 μg/ml — 76 —

7723330 methyl 4-({[3- (1,3- benzodioxol- 5-ylmethyl)- 2,5-dioxo-1-phenyl-4- imidazol idinyl]acetyl} amino) benzoate 965 26 >10 μg/ml — 77IV

7199725 4-(3,4- dihydro-2(1H)- isoquinolinyl- methyl)-N-[2-(1-pyrrolidinyl carbonyl) phenyl] benzamide 987 25 >10 μg/ml — 78 —

6987235 9-{3-chloro-4- [(4-methyl benzyl)oxy] phenyl}-10- ethyl-3,4,6,7,9,10- hexahydro- 1,8(2H,5H)- acridine dione 680 25 >10 μg/ml —79 _—

7140931 ethyl 1-(4- {[(3,4-dimethyl phenyl) (methyl sulphonyl) amino]methyl} benzoyl)-4- piperidine carboxylate 14 24.5 >10 μg/ml — 80 —

7787455 methyl 4-{[N- (3-methoxy phenyl)-N- (phenyl sulphonyl) glycyl]amino} bonzoate 407 20 >10 μg/ml — 81 III

7660465 N-{[5-({2-[(4- bromo-2,3- dimethyl phenyl) amino]-2- oxoethyl}thio)-4-ethyl- 4H-1,2,4- triazol-3- yl]methyl}-4- chloro benzamide 88120 ~7 μg/ml 3.2 μM 82 II

7661751 4-(2,3- dihydro-1,4- benzodioxin- 6-yl carbonyl)- 3-hydroxy-1-[3-(4- morpholinyl) propyl]-5-(4- pyridinyl)-1,5- dihydro-2H-pyrrol-2-one 951 19 ~10 μg/ml — 83 I

7722914 N-(4-ethoxy phenyl)-2-{1- (4-methoxy phenyl)-3-[2- (4-morpholinyl) ethyl]-5-oxo-2- thioxo-4- imidazolidinyl} acetamide 806 19~7 μg/ml 15.6 μM 84 IV

7745040 5-(3-bromo phenyl)-3- hydroxy-4-[(7- methoxy-1- benzo furan-2-yl) carbonyl]-1-[2- (4- morpholinyl) ethyl]-1,5- dihydro-2H-pyrrol-2-one 313 18 ~10 μg/ml — 85 I

7735385 1-[3-(diethyl amino)propyl]- 3-hydroxy-4- [(7-methoxy-1-benzofuran- 2-yl) carbonyl]-5- (2-pyridinyl)- 1,5-dihydro-2H-pyrrol-2- one 21 17 ~10 μg/ml — 86 I

7756003 1-(4-{[(4,6- dimethyl-2- pyrimidinyl) thio]acetyl}-1-piperazinyl)-4- (4-methyl phenyl) phthalazine 361 11 ~5 μg/ml — 87 —_(—)CB ref: ChemBridge reference; Rank: rank of each molecule in the list ofthe 1000 best molecules after in silico docking; In vit. inhib.: meaninhibition % obtained with each molecule for the displacement of thebinding of scFv G4G11 with Syk in vitro in the ADA method; Degran. IC50:concentration inhibiting mast cell degranulation by 50%; Kd:dissociation constant with respect to Syk measured in vitro byspectrofluorometry No.: number assigned by the inventors Gp: groups towhich the molecules belong.

Example 3 Materials and Methods

1) Chemical products and antibodies. A chemical bank of 3000 molecules(a varied subset) was acquired from ChemBridge, Inc. (San Diego,Calif.). Stocks of solutions of small molecules were prepared at a rateof 10 mM in DMSO (dimethylsulphoxide), except for C-13 (methyl2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}benzoate,Chem Bridge ID No. 6197026) prepared in DMF (dimethylformamide). Unlessspecified otherwise, all the reagents were supplied by Sigma.Dinitrophenyl (DNP) hapten was acquired from Calbiochem. SepharoseGammaBind G and all the secondary antibodies were supplied by GE HealthAmersham Biosciences. The anti-Syk, anti-Lyn, anti-Btk, anti-PLC-γ1,anti-PLC-γ2, anti-LAT, anti-SLP-76, anti-p38, anti-JNK, anti-Vav,anti-Akt1 and 9E10 antibodies conjugated with HRP were acquired fromSanta Cruz Biotechnology. The anti-phospho-p44/42 MAP Kinase,anti-p44/42 MAP Kinase, anti-phospho-p38, anti-phospho-JNK,anti-phospho-Akt1 antibodies were acquired from Cell Signaling. Theanti-phospho-LAT and anti-phospho-PLC-γ1 antibodies were supplied byBiosource and the anti-phospho-SLP-76 antibodies by BD Pharmingen. The4G10 anti-phosphotyrosine monoclonal antibody was acquired from UpstateBiotechnology.

2) ADA method: ELISA type high-speed molecule screening test, based onantibody displacement (WO 2005106481). The recombinant fusion proteinGST:Syk 6-242⁸ comprising the residues 6 to 242 of murine Syk tyrosinekinase protein illustrated in FIG. 8B (SEQ ID No. 4) was immobilised onan ELISA plate at a final concentration of 10 μg ml⁻¹. For the screeningof the chemical molecule bank, the small molecules, diluted in PBS at afinal concentration of 10 μM were added to the wells for one hour atambient temperature, before adding the fragment scFv G4G11 at a finalconcentration of 100 nM for an additional hour. The binding of G4G11with Syk was assessed by adding the 9E10 monoclonal antibody conjugatedwith HRP detecting the amino acid sequence EQKLISEEDLN of human c-mycprotein located at the C-terminal end of the scFv fragment. To generateSyk mutants, targeted mutagenesis was used on GST:Syk 6-242 protein andthe binding of G4G11 with the mutants was assessed in the presence of 5μM of C-13.

3) Cells, culture conditions and functional tests. Anti-DNP 2682-I mousemonoclonal antibody was used as the culture supernatant of hybridomascontaining 1 μg/ml of IgE. Femoral bone marrow cells were sampled andcultured in Opti-MEM medium (Gibco) supplemented with 10% foetal calfserum and 4% X63 transfectant supernatant secreting murine IL-3. RBL-2H3(ATCC) leukaemic rat basophil cells were maintained in a single-layerculture in RPMI 1640 medium supplemented with 10% foetal calf serum(Gibco). Measurements of β-hexosaminidase released by the RBL-2H3 cellswere performed as described previously⁷, except that, after 12-16 hoursof incubation with anti-DNP IgE (0.5 μg/ml), the cells were incubatedfor 90 min at 37° C. in RPMI medium supplemented with the specifiedconcentrations of C-13 or DMF (0.25%). The cells were stimulated for 45min with DNP-BSA (50 ng ml⁻¹) or ionomycin (1.5 μM). The BMMC cells wereincubated for one hour at 37° C. with anti-DNP IgE (100 ng/ml). Theywere then incubated with C-13 (3 μM) or DMF (0.3%) for 3 hours at 37°C., and stimulated with varied concentrations of DNP-BSA. The level ofβ-hexosaminidase released was measured 10 min later and the TNF-αtitration was performed by means of a cytotoxicity test on L929 cells asdescribed previously¹⁰, 3 hours after stimulation. The resultsillustrated in FIG. 3 are representative of three independentexperiments.

4) Immunoprecipitations, in vitro kinase assays and immunodetection. Allthe experiments were conducted as described previously⁷, except that,before stimulation with DNP-BSA (50 ng ml⁻¹, 3 min), the RBL-2H3 cellswere incubated for 90 min at 37° C. in RPMI medium supplemented with thespecified concentrations of C-13 or DMF. The cells were solubilised inDOC modified lysis buffer (1% NP-40, 0.25% sodium deoxycholate, 0.1% SDSin PBS buffer supplemented with protease and phosphatase inhibitors) andthe protein concentration was determined (BCA Protein Assay, PIERCE).For immunoprecipitations, cell lysates, non-stimulated and stimulatedwith IgE/DNP were incubated with preformed complexes of antibodies andSepharose GammaBind G, and the in vitro kinase activity of Syk, Btk andLyn immunoprecipitates were examined. Before SDS-PAGE gel separation thelysates or immunoprecipitates were prepared by adding SDS sample buffer(60 mM Tris, pH 6.8, 2.3% SDS, 10% glycerol, 0.01% bromophenol blue).The proteins were transferred onto a nitrocellulose membrane (Schleicher& Schuell), and detected using suitable antibodies and thechemoluminescence system improved (ExactaCruz, Santa CruzBiotechnology).

5) Flow cytometry analysis of level and calcium mobilisation and FcεRImembrane expression. The intracellular free calcium concentration wasdetermined by previously charging 1×10⁶ cells with 5 mM of Fluo-3 AM(Molecular Probes, Invitrogen) in the presence of 0.2% Pluronic F-127for 30 min at ambient temperature. Prior to stimulation with DNP-BSA orionomycin, the cells were treated for 90 min at 37° C. in RPMI mediumsupplemented with C-13 or DMF (0.25%), and the intracellular freecalcium concentration was measured with a flow cytometer (BecktonDickinson). For the FcεRI surface expression evaluation, the cells wereincubated for 2 hours at 37° C. with anti-DNP IgE. Membrane-bound IgEwas detected using biotinylated anti-mouse Ig, and streptavidineconjugated with Fitc.

6) Anaphylaxis induction. Female BALB/c mice (aged 6-8 weeks) wereacquired from Charles River and kept at the IRCM animal house underpathogen-free conditions. The Ig-dependent passive systemic anaphylaxis(PSA) and passive cutaneous anaphylaxis (PCA) protocols were conductedas described previously¹¹. Briefly, the mice received, by intravenousinjection, 100 μg of IgE (SPE-7, Sigma) in 200 μl of PBS for PSA, or, byintradermal injection, 25 ng of IgE in 10 μl of PBS for PCA, and werestimulated 24 hours later by means of an intravenous injection of 1 mgof DNP-KLH in 2% of Evans blue. C-13, a non-relevant chemical moleculeor the vehicle were administered 1 hours before stimulation, eitherorally (PSA) in 200 μl of 1% carboxymethylcellulose, or locally in theear by means of intradermal injection (PCA) in an acetone/olive oilmixture (4:1). The animals were sacrificed 20 min after stimulation. Theears were removed, ground and Evans blue was extracted after overnightincubation in formamide at 80° C. For the temperature measurements inPSA, C-13 (100 mg/kg) or the vehicle were administered orally, 3 hoursprior to stimulation performed in the absence of Evans blue. Thetemperature was measured using an electronic thermometer with a rectalprobe (YSI, Yellow Springs, Ohio) before stimulation and for 60 minutesafterwards, prior to sacrifice. The absorbance was measured at 610 nm.The experiments were conducted with 4-5 mice per condition. The dataillustrated in FIG. 4 are representative of three different experiments.

7) Structural studies. The three-dimensional cavities liable to bepharmaceutical targets were predicted using Q-SiteFinder¹² and ICM¹³.The molecule C-13 was docked using LigandFit¹⁴ and Surflex¹⁵. The first20 positions were analysed and a consensus position is given in FIG. 1A.The images were generated with PyMol.

8) Peritonitis (FIG. 5). Syk-dependent peritoneal neutrophil recruitmentwas induced in 8-week old female BALB/c mice as described in³⁶ byintravenous injection of 4 μg of Bordetella pertussis toxin (donated byDr D. Raze, Inserm, Lille, France) and 2 hours later by intraperitonealinjection of 4% thioglycollate in sterile water. A peritoneal lavage wasperformed with 5 ml of PBS 4 hours later and the total number ofneutrophils was determined after labelling with anti-Gr1 conjugated withAPC (Becton-Dickinson) and flow cytometry analysis. C-13 (100 mg/kg) inCMC or the vehicle alone was administered orally one hour prior toinjecting Bordetella pertussis toxin.

9) In vitro B Lymphocyte purification and proliferation. Spleen Blymphocyte cells were purified from 8-week old female BALB/c mice onmagnetic beads by negative selection using micro-beads coated with CD43and LS columns (Miltenyi Biotec) as described previously³⁷. After twohours of incubation with variable concentrations of C-13 or the vehicle,50,000 cells were cultured for 48 hours in 96-well plates in thepresence of absence of 10 μg/ml of donkey anti-mouse IgM F(ab′)₂fragment (Jacson Immunoresearch). Alamar blue (Serotec) was added to thecultures 24 prior to measuring the reduced versus oxidised forms of thereagent at 570 and 620 nm, in accordance with the manufacturersinstructions.

10) Antibody production (FIG. 5C). This experiment was conducted asdescribed in³⁸. Eight-week old female BALB/c mice received an oral doseof C-13 (150 mg/kg) in 1% CMC or the vehicle alone, were immunised 3hours later by an intraperitoneal injection of 10 μg oftrinitophenyl-keyhole limpet haemocyanin (TNP-KLH) in Rehydragel alum(Reheiss). The serum was collected before immunisation and on day 12.The antigen-specific immunoglobulin levels were measured by means ofELISA with 10 μg/ml of plate-bound TNP-OVA (Biosearch technologies) asthe capture agent. The IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA levels weremeasured using samples diluted to 1:5000 using goat antibodiesconjugated with peroxidase and isotype-specific (SouthernBiotechnology), whereas the IgE levels were measured using samplesdiluted to 1:50 using biotinylated anti-mouse IgE rat antibodies (BectonDickinson) and streptavidine conjugated with peroxidase (R&D Systems).After incubation with TMB substrate, the optical density (OD) wasmeasured at 450 nm.

11) Statistical analyses. The mean numeric data are expressed asmeans±standard deviations (SD). Students t test was used to determinethe statistical significance of the differences between groups.

12) C-13 toxicity test on BMMC cells. To assess the potential toxiceffect of C-13 on mast cells, BMMC cells were incubated for 5 days at37° C. in the presence of 2.5 μM or 5 μM of C-13, or 0.25% DMF(corresponding to the DMF concentration used with 5 μM of C-13) underthe same conditions as for functional tests. Double labelling withAnnexin-V and Propidium Iodide demonstrated the level of BMMC cellviability after 3 hours and after 5 days. The viability of the BMMCcells treated under the same conditions with Staurosporine was measuredunder the same conditions.

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1. A compound which binds with Syk tyrosine kinase protein on athree-dimensional cavity comprising the Arginine residue situated inposition 68 and the two glutamic acid residues situated in positions 121and 155 of human Syk protein, the sequence of which is set forth in SEQID No. 1, and capable of inhibiting by at least 10% in vitro binding of(i) antibody fragment G4G11 (SEQ ID No. 2), or (ii) an antibody orantibody fragment which binds with human Syk tyrosine kinase protein onan epitope comprising at least one of residues 65 to 74 of the aminoacid sequence of human Syk tyrosine kinase protein represented by thesequence SEQ ID No. 1, or (iii) an antibody or antibody fragment whichbinds with human Syk tyrosine kinase protein and inhibiting by at least10% the binding of antibody fragments G4G11 with human Syk tyrosinekinase protein (SEQ ID No. 1), with human Syk tyrosine kinase protein orwith any of the variants thereof in animals, for the prevention ortreatment of a condition dependent on a metabolic pathway involving Sykin humans or animals, where said compound (C-13) has the formula

or an organic compound functionally equivalent to molecule C-13 having amolecular weight between 50 and 2500 Dalton, selected from compoundshaving any of the following formulae:

where R1 is an unsubstituted or substituted aromatic group, or anunsubstituted or substituted heterocycle comprising at least one S, O orN atom; R2 is an unsubstituted or substituted aromatic group, anunsubstituted or substituted heterocycle, an unsaturated or saturatedcarbon chain comprising an amine group, an unsaturated or saturatedcarbon chain comprising an unsubstituted or substituted aromatic groupor an unsaturated or saturated carbon chain comprising an unsubstitutedor substituted heterocycle comprising at least one S, O or N atom; R3 isan unsubstituted or substituted phenyl, 2-pyridinyl, 3-pyridinyl or4-pyridinyl group; or

where n=0 or 1; n′=0 or 1; R4 is an unsaturated or saturated carbonchain comprising 1 to 5 carbon atoms, unsubstituted or substituted withan aromatic group; R5 is an unsubstituted or substituted aromatic groupor an unsubstituted or substituted amine group; R6 is a hydrogen atom,an alkoxy group, an alkyl group or a halogen; R7 is a hydrogen atom, analkoxy group, an alkyl group or a halogen; R8 is a hydrogen atom, analkoxy group, an alkyl group or a halogen; or

where m=0, 1 or 2; R9 is a hydrogen atom and R10 is an unsubstituted orsubstituted phenyl group, or R9 and R10 are part of the sameunsubstituted or substituted heterocycle, or R9 and R10 are part of thesame unsubstituted or substituted aromatic group; R11 is a hydrogenatom, an alkoxy group or an alkyl group; R12 is a hydrogen atom, analkoxy group or an alkyl group; R13 is a hydrogen atom or an alkyl oralkoxy group; R14 is a hydrogen atom or an alkyl or alkoxy group;

or where A is an oxygen or sulphur atom; R15 is an unsaturated orsaturated carbon chain comprising 1, 2 or 3 carbon atoms, unsubstitutedor substituted by an unsubstituted or substituted aromatic group, anunsubstituted or substituted heterocycle or an amine group belonging tounsubstituted or substituted heterocycle; R16 is a hydrogen atom, ahalogen or an alkoxy group; R17 is a hydrogen atom, an alkoxy group oran acetoxy group; or

or a stereo-isomer, racemate or pharmacologically acceptable salt ofC-13 or said equivalent compound.
 2. A compound according to claim 1,wherein said compound binds with Syk tyrosine kinase protein at a sitelocated outside the catalytic domain thereof.
 3. A compound according toclaim 1, wherein said antibody or antibody fragment binds with human Syktyrosine kinase protein on an epitope comprising at least 5 of residues65 to 74 of the amino acid sequence of human Syk tyrosine kinase proteinillustrated by SEQ ID No.
 1. 4. A compound according to claim 1, whereinsaid three-dimensional cavity further comprises the Serine residuesituated in position 9, the Glutamine residue situated in position 43,the Phenylalanine residue situated in position 51, the Isoleucineresidue situated in position 66, the Glutamate residues situated inposition 67 and 69, the Leucine residue situated in position 70, theAsparagine residue situated in position 71, the Glycine residue situatedin position 72, the Threonine residue situated in position 73, theTyrosine residue situated in position 74 and the Alanine residuesituated in position 75 of human Syk protein, the sequence of which isillustrated by SEQ ID No.
 1. 5. A compound according to claim 1 whereinsaid compound is used for the prevention or treatment of type Ihypersensitivity reactions.
 6. A compound according to claim 5, whereinsaid compound inhibits IgE-dependent mast cell degranulation.
 7. Acompound according to claim 6, wherein said compound is capable ofinhibiting, by 50% in vitro, mast cell degranulation at a concentration(IC50) between 1 ng/ml and 1 mg/ml.
 8. A compound according to claim 5,wherein said the metabolic pathway involving Syk is a mast cell orbasophil activation pathway.
 9. Molecule A compound according to claim5, wherein said condition is allergic asthma, allergic conjunctivitis,allergic rhinitis, anaphylaxis, angioedema, urticaria, eosinophilia oran allergy to an antibiotic.
 10. A compound according to claim 5,wherein said compound has no effect on the metabolic pathways involvinghuman Syk protein (SEQ ID No. 1) other than those giving rise to mastcell degranulation and/or type I hypersensitivity reactions.
 11. Acompound according to claim 10, wherein said compound has no effect onthe antibody response which follows immunisation by a thymus-dependentantigen or on neutrophil-dependent Syk recruitment.
 12. A compoundaccording to claim 1, wherein said metabolic pathway involving Syk is aB lymphocyte, T lymphocyte, neutrophil, eosinophil, NK cell, platelet,erythrocyte, osteoclast, epithelial cell or cancer cell activationpathway.
 13. A compound according to claim 12, wherein said condition isrheumatoid arthritis, an autoimmune disease, inflammation or cancer. 14.A compound according to claim 1, wherein said compound is used incombination with a further therapeutic molecule.
 15. (canceled) 16.(canceled)
 17. A compound according to claim 1, wherein said compound isintended to be administered by the oral, sublingual, nasal, ocular,local, intravenous, intraperitoneal, subcutaneous route, by aerosol orby inhalation.
 18. A compound according to claim 1, wherein saidcompound is intended to be administered to adult, child or newborn humanpatients.
 19. A compound according to claim 1, wherein said compound isintended to be administered at doses between 0.01 mg/kg and 200 mg/kg.20. A compound according to claim 19, wherein said equivalent compoundis selected from compounds having any of the following formulae: formula(I) where R1 is a phenyl group, unsubstituted or substituted by an F orCl atom, a methyl or ethyl group, an N,N-dimethyl-sulphonamide or twogroups selected from the methyl, ethyl, hydroxy, methoxy or ethoxygroups, or a group

a furan group unsubstituted or substituted by a methyl, ethyl, hydroxyl,methoxy or ethoxy group, a thiophene group unsubstituted or substitutedby a methyl, ethyl, hydroxy, methoxy or ethoxy group; R2 a group

where R21 and R22 are carbon atoms each belonging to an alkyl chaincomprising 1, 2 or 3 carbon atoms, or both belonging with the nitrogenatom with which they are bound to the same unsaturated or saturatedheterocycle also comprising an oxygen atom or a second nitrogen atom, ora group

where R23 and R24 are carbon atoms each belonging to an alkyl chaincomprising 1, 2 or 3 carbon atoms, or both belonging with the nitrogenatom with which they are bound to the same unsaturated or saturatedheterocycle also comprising an oxygen atom or a second nitrogen atom, ora group

and R3 is a non-substituted 2-pyridinyl, 3-pyridinyl or 4-pyridinylgroup, a phenyl group unsubstituted or substituted by a benzoxy group,and/or by a hydroxyl group, and/or by a methyl group, and/or by an ethylgroup, and/or by a propyl group, and/or by one or two Br, F or Cl atoms,and/or by one to three hydroxyl, methoxy or ethoxy groups. or formula(II) where R4 is an unsaturated or saturated carbon chain comprising 1,2 or 3 carbon atoms; R5 is a phenyl group or a secondary amine groupunsubstitued or substituted by an unsubstituted or substituted phenylgroup, or by a group

R6 is a hydrogen or chlorine atom or a methyl, ethyl, hydroxyl, methoxyor ethoxy group; R7 is a hydrogen or chlorine atom or a methyl, ethyl,hydroxy, methoxy or ethoxy group; R8 is a hydrogen or chlorine atom or amethyl, ethyl, hydroxy, methoxy or ethoxy group. or the formula (III)where R9 is a hydrogen atom and the group R10 is an unsubstituted orsubstituted phenyl group, or the groups R9 and R10 belong to the sameunsubstituted or substituted heterocycle comprising 2 nitrogen atoms and4 carbon atoms; R11 is a hydrogen atom or methyl, ethyl, hydroxy,methoxy or ethoxy group; R12 is a hydrogen atom or methyl, ethyl,hydroxy, methoxy or ethoxy group; R13 is a hydrogen atom or methyl,ethyl, hydroxy, methoxy or ethoxy group; R14 is a hydrogen atom ormethyl, ethyl, hydroxy, methoxy or ethoxy group; or formula (IV) where Ais an Oxygen or Sulphur atom; R15 is a group

R16 is a hydrogen or chlorine atom or a methyl, ethyl, hydroxy, methoxyor ethoxy group; R17 is a methyl, ethyl, hydroxy, methoxy, ethoxy,acetoxy, methoxycarhonyl or ethoxycarbonyl group.
 21. A compoundaccording to claim 20, wherein said equivalent compound is selected fromthe following:


22. A compound according to claim 1, wherein the in vitro affinity ofsaid compound for Syk protein is less than 25 μM.
 23. A compositioncomprising a compound according to claim 1 and a pharmacologicallyacceptable excipient for the prevention or treatment of a conditiondependent on a metabolic pathway involving Syk in humans or animals. 24.(canceled)
 25. A method for identifying an organic compound having amolecular weight between 50 and 2500 Dalton binding with Syk tyrosinekinase protein and capable of inhibiting by at least 10% in vitrobinding of (i) antibody fragment G4G11 (SEQ ID No. 2), or (ii) anantibody or antibody fragment which binds with human Syk tyrosine kinaseprotein on an epitope comprising at least one of residues 65 to 74 ofthe amino acid sequence of human Syk tyrosine kinase protein representedby the sequence SEQ ID No. 1, or (iii) an antibody or antibody fragmentwhich binds with human Syk tyrosine kinase protein and inhibiting by atleast 10% the binding of antibody fragment G4G11 with human Syk tyrosinekinase protein (SEQ ID No. 1), with human Syk tyrosine kinase protein orwith any of the variants thereof in animals, comprising at least thefollowing steps: a) screening, from a bank of candidate organiccompounds having a molecular weight between 50 and 2500 Da, those liableto bind with Syk protein on the three-dimensional binding cavity on theSyk protein of a compound selected from the molecules having formulaC-13, I, II, III, IV or 1 to 87 as illustrated above; b) selecting fromthe compounds identified in a) those capable of inhibiting by at least10% in vitro the binding of the antibody or antibody fragment (i) or(ii) with Syk protein.
 26. (canceled)
 27. (canceled)
 28. (canceled) 29.(canceled)
 30. (canceled)